Importance of calcium and potassium currents in human lens epithelial cells (hLEC) and the effect of the calcium channel blocker mibefradil

Background: To prevent posterior capsule opacification (PCO), we followed the hypothesis that calcium channel blockers (antagonists) interfere with integrin signaling and block cell adhesion in lens epithelial cells (LEC). In primary human LEC we found that the T channel antagonist mibefradil induces apoptosis which was accompanied with cell shape changes and loss of cell adhesion. Although T-type calcium channels are substantially present in membranes of freshly dispersed primary cultured hLEC and calcium currents are inhibited by mibefradil at concentrations of 10-8 M, the antiproliferative site of action of this drug remains unclear, since this feature is observed at concentrations 200-fold higher than that for calcium channel blockade. Methods: Epithelial cells of the human lens were dispersed by enzymatic treatment, recordings of membrane currents were performed using patch clamp technique in the whole cell configuration. Westernblot analysis was used for protein detection. Results: Total current elicited on depolarizing voltage steps from a holding potential of-80 mV was composed of inward (calcium) and outward (potassium) current. Outward current could be inhibited mostly by intracellular application of cesium ions. Currents in inward direction were activated fast (< 2 ms) and inactivated during the following 20 ms. They were characterized as calcium currents since the known calcium channel inhibitor nifedipine blocked these currents in a concentration-dependent manner. Using potassium in the pipette (145 mM) as main charge carrier, additionally a noninactivating potassium current and a voltage- and time-dependent potassium current which slowly inactivated (Kv) were observed (control). Adding mibefradil in concentrations from 10-6 M - 10-5 M to the bath solution, the inwardly directed and the non-inactivating current component were inhibited concentration-dependent. The Kv component was affected in a similar way, however, this component showed an increased inactivation behavior after application of mibefradil in the named concentrations. Conclusion: The total effects of mibefradil are significant for the calcium homeostasis since calcium current itself is inhibited but, moreover, the membrane is permanently depolarized up to 20 mV by the drug. Both effects may contribute to the observed reduced cell adhesion during mibefradil treatment. (Less)